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Vol. 17, Issue 10, 4282-4299, October 2006

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Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

Wenyu Wen^*,, Lu Chen^,, Hao Wu^*, Xin Sun, Mingjie Zhang^*, and David K. Banfield

Departments of *Biochemistry and Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Special Administrative Region of China

Submitted February 13, 2006; Revised June 20, 2006; Accepted July 11, 2006
Monitoring Editor: Benjamin Glick

Using nuclear magnetic resonance spectroscopy, we establish that the N-terminal domain of the yeast vacuolar R-SNARE Nyv1p adopts a longin-like fold similar to those of Sec22b and Ykt6p. Nyv1p is sorted to the limiting membrane of the vacuole via the adaptor protein (AP)3 adaptin pathway, and we show that its longin domain is sufficient to direct transport to this location. In contrast, we found that the longin domains of Sec22p and Ykt6p were not sufficient to direct their localization. A YXX-like adaptin-dependent sorting signal (Y³¹GTI³⁴) unique to the longin domain of Nyv1p mediates interactions with the AP3 complex in vivo and in vitro. We show that amino acid substitutions to Y³¹GTI³⁴ (Y31Q;I34Q) resulted in mislocalization of Nyv1p as well as reduced binding of the mutant protein to the AP3 complex. Although the sorting of Nyv1p to the limiting membrane of the vacuole is dependent upon the Y³¹GTI³⁴ motif, and Y31 in particular, our findings with structure-based amino acid substitutions in the mu chain (Apm3p) of yeast AP3 suggest a mechanistically distinct role for this subunit in the recognition of YXX-like sorting signals.

These authors contributed equally to this work.

Address correspondence to: David K. Banfield (bodkb{at}ust.hk)

Abbreviations used: AP, adaptor protein; ER, endoplasmic reticulum; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl) pyridinium dibromide; GFP, green fluorescent protein; GST, glutathione S-transferase; LD, longin domain; SNARE, soluble N-ethylmaleimide-sensitive factor attachment receptor protein.

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